The actin cytoskeleton is essential for cell viability and plays critical roles in cell shape, adhesion, and motility. Normal physiological functions associated with respiration, circulation, locomotion, and digestion expose cells to mechanical stress that can deform or damage cytoskeletal elements. A major challenge in cell biology is to understand how living cells monitor the integrity of the actin cytoskeleton and make repairs to maintain structure and function. This proposal builds on our recent discovery of a novel homeostatic mechanism by which cells maintain the structural integrity of the actin cytoskeleton in the face of mechanical stress. Actin stress fibers undergo spontaneous cycles of thinning and repair that can be induced by the direct application of physical force. A Stress Fiber-Remodeling, Repair, and Reinforcement (SF-R3) complex that includes zyxin, Ena-VASP proteins, and 1-actinin, has been identified. The SF-R3 complex is rapidly recruited to regions of stress fiber thinning, or damage, where it facilitates actin remodeling and stress fiber reinforcement. The SF-R3 complex reduces the incidence of catastrophic stress fiber breaks and restores the capacity of stress fibers to transmit force via the focal adhesions to the underlying substrate. Genetic studies have revealed that zyxin is essential for recruitment of the repair complex to sites of stress fiber strain. The proposed research will employ a transdisciplinary approach that incorporates genetic models, biochemistry, quantitative fluorescence imaging, and traction force microscopy to probe the mechanism and physiological impact of the actin repair machinery. The first aim is to define the molecular signals that mark regions of stress fiber damage and to determine how the SF-R3 complex is targeted, with a high degree of spatial precision, to those sites. The second aim is to dissect the mechanism by which components of the repair complex contribute to actin reinforcement and restoration of contractile function. The third aim will employ the murine lung as a model for studying the physiological role of the SF-R3 complex in an organ exposed to mechanical stress, by testing mice harboring mutations that affect components of the repair machinery for their response to mechanical ventilation, a clinically important intervention that often leads to pathophysiological consequences such as loss of alveolar- capillary barrier function. The experimental plan is innovative because it probes the fundamental mechanism and physiological impact of a novel machinery for actin cytoskeletal maintenance and repair that is activated by mechanical stress. The proposed research is significant because a molecular understanding of how cells remodel actin in response to mechanical stress may suggest strategies for therapeutic intervention for pathological conditions driven by excess mechanical stress, such as ventilator-induced lung injury.